Antiviral composition and method of use

ABSTRACT

Disclosed herein is a method of reducing the risk of a rhinovirus infection, the method includes administering to a subject a composition comprising iota-carrageenan in an antiviral effective amount as an active antiviral ingredient.

This is a Division of application Ser. No. 12/312,926 filed Aug. 3,2009, which in turn is a National Stage Application ofPCT/EP2007/010512, filed Dec. 4, 2007, which in turn is acontinuation-in-part of U.S. patent application Ser. No. 11/567,078,filed Dec. 5, 2006 now abandoned. The disclosure of the priorapplications is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention is in the field of immunology and antiviral agentsand relates to a pharmaceutical composition comprising carrageenan as anactive antiviral ingredient and to applications thereof in theprevention or therapeutic treatment of rhinovirus infections.

BACKGROUND OF THE INVENTION

Picornaviruses represent a very large virus family of small ribonucleicacid-containing viruses responsible for many serious human and animaldiseases. Picornaviruses include four major groups: enteroviruses,rhinoviruses, cardioviruses and apthoviruses.

The human rhinoviruses consist of at least 100 serotypes and are theprimary causative agents of the common cold. Because of the large numberof serotypes, development of a vaccine is problematic; antiviral agentsmay therefore be the best approach to treatment. Rhinoviruses arecomposed of a surrounding capsid, which contains four viral proteinsVP1, VP2, VP3 and VP4. Proteins VP1, VP2 and VP3 are organized into 60repeating protameric icosahedral units. These are thought to be thecause of antigen diversity associated with these viruses.

Rhinovirus (HRV) infections lead to the common cold with symptoms suchas fever, cough, and nasal congestion. HRV infection is the second mostfrequently recognized agent associated with pneumonia and bronchiolitisin infants and young children and commonly causes exacerbations ofpre-existing airways disease in those with asthma, chronic obstructivepulmonary disease or cystic fibrosis. HRV infection is associated withone-third to one-half of asthma exacerbations depending on age and islinked to asthma hospitalizations in both adults and children.

Present treatment approaches include the application of rhinovirusspecific RNA, as disclosed in the DE 19825395, which binds to the canyonregion of the capsid, which is necessary to host receptor (e.g. ICAM-1,intercellular adhesion molecule-1) binding and cell infection.

Another method comprises the administration of soluble ICAM-1 proteinsor derivatives of ICAM-1 as disclosed in the U.S. Pat. No. 6,326,004 andthe U.S. Pat. No. 6,096,862 (tICAM-1, truncated ICAM-1) to neutralizeviral particles (virions).

Chemical compounds with antiviral activity against rhinoviruses aredisclosed in EP 0523803.

Rhinovirus symptoms are caused by an overly or unspecific reaction ofthe immune system. Therefore common treatment forms of a rhinovirusinfection include administration of analgesics such as aspirin oracetaminophen/paracetamol, as well as localized versions targeting thethroat (often delivered in lozenge form), nasal decongestants whichreduce the inflammation in the nasal passages by constricting localblood vessels, cough suppressants (which work to suppress the coughreflex of the brain or by diluting the mucus in the lungs), andfirst-generation anti-histamines such as brompheniramine,chlorpheniramine, and clemastine (which reduce mucus gland secretion andthus combat blocked/runny noses but also may make the user drowsy).

Sulphated polysaccharides including carrageenans are known in the artfor their antiviral efficacy. In a most interesting review, Gonzalez M.E. et al. (1987, Antimicrob. Agents Chemother. 31, 1388-1393) report anantiviral efficacy of different sulphated polysaccharides includingiota-carrageenan against several animal viruses. Iota-carrageenan showedantiviral activity against the enveloped viruses HSV-1, HSV-2, SemlikiForest virus (SFV), vaccinia virus and African swine fever virus (ASF)and against the naked encephalomyocarditis (EMC) virus. Iota-carrageenanhad no effect on the enveloped viruses vesicular stomatitis virus (VSV)and measles virus and on the naked viruses polio virus type 1 andadenovirus type 5.

US 2003/181415 A discloses antiviral activity of sulfatedpolysaccharides such as cellulose sulfate, against various envelopedviruses and in particular herpes simplex virus (HSV), Papilloma virusesand HIV.

WO 2005/004882 A discloses therapeutic treatment of viral infections,excluding rhinovirus infection, with sulphated polysaccharides such ascarrageenans.

US 2005/171053 A1 discloses the use of lambda-carrageenan for inhibitingthe spread of a sexually transmitted infection including HIV-1infection.

Yamada et al. (1997, Carbohydrate Polymers, Appl. Scien. Publishers 32,51-55) disclose an anti-HIV-1 activity of lambda-, kappa- andiota-carrageenan and their sulphated derivatives.

S. F. Tischer et al. (2006, Carbohydrate Polymers, Appl. Scien.Publishers 63, 459-465) disclose an activity of carrageenan isolatedfrom Meristiella gelidium against herpes simplex and dengue virus.

Carlucci et al. (2004, Antiviral Research, Elsevier Science BV. 64,137-141) disclose a protective effect of lambda-carrageenan on genitalherpes simplex virus infection in mice.

Pujol et al. (2006, Planta Medica 72, 121-125) disclose an antiviralactivity of a carrageenan isolated from Gigartina skottsbergii againstintraperitoneal murine herpes simplex virus infection.

The term “carrageenan” is frequently used a collective term for linearsulphated galactose-based polysaccharides extracted from seaweed(rhodophyceae). It is mostly used as a thickener, gelling agent,stabilizer or emulsifier in pharmaceutical and food products. Thereexist more than 10 different carrageenans depending on the seaweed genusfrom which they are extracted. The three main types are iota-, kappa-and lambda-carrageenan, which differ slightly in their structure anddegree of sulphatation. Iota-carrageenan is a soft-gel forming sulphatedgalactan predominantly extracted from red seaweed Gigartina stellata andChondrus crispus. Kappa-carrageenan yields strong, rigid gels and ispredominantly produced from Kappaphycus cottonii. Lambda-carrageenan,which is the most common form, is frequently used to thicken dairyproducts.

Despite the long known antiviral activity of some carrageenans againstviruses such as, e.g. HIV or HSV, the mechanism of how carrageenansexhibit antiviral activity still needs clarification.

In the light of the above, the present invention now provides for acarrageenan-based antiviral composition suitable in the prophylactic ortherapeutic treatment of rhinovirus infections (rhinitis).

Experiments leading to the present invention have surprisinglydemonstrated that in spite of possible reservations in the art selectedcarrageenans exert antiviral activity against rhinovirus infections(rhinitis), with iota-carrageenan yielding the best results.

DESCRIPTION OF THE INVENTION

Therefore, the present invention in its first embodiment relates to theuse of carrageenan as an active antiviral ingredient in the manufactureof a pharmaceutical composition for the prophylactic or therapeutictreatment of a rhinovirus infection.

The term “active antiviral ingredient” used herein refers to acarrageenan compound that when applied in an effective dose or amountinterferes, directly or indirectly or both directly and indirectly, withthe rhinovirus infection cycle of a eukaryotic cell, more specificallywith at least one part of the rhinovirus infection cycle selected fromthe group consisting of virus penetration of a eukaryotic cell, virusreplication in a eukaryotic cell, virus assembly, and virus release fromthe infected eukaryotic cell. It also encompasses any effect inunspecifically inhibiting a virus titer increase or in unspecificallyreducing a virus titer level in a eukaryotic or mammalian host system.The term further refers to a compound that has prophylactic efficacy inthat it protects from, at least to some extent, or reduces thelikelihood of coming down with a viral infection.

The present pharmaceutical composition may thus be administered beforeor after the onset of a viral infection. The term “prophylaxis” or“prophylactic treatment” as used herein relates to the administration ofthe present pharmaceutical composition in order to protect from, atleast to some extent, or reduce the risk of falling ill with a viralinfection.

The term “therapy” or “therapeutic treatment” as used herein relates tothe administration of the present pharmaceutical composition to avirus-infected individual in order to alleviate the pathological impactof the infection, including reduction in severity and/or frequency ofemerging symptoms, or elimination of such symptoms, remediation ofpossible injuries caused by or associated with such viral infection, andincluding inhibition or prevention of secondary viral, bacterial, fungalor any other kind of microbial infection.

The collective term “carrageenan” as used hereinafter refers to amixture of at least two of iota-kappa- and lambda-carrageenan homo- orheteropolymers, i.e. to a mixture of iota- and lambda-carrageenan, amixture of iota- and kappa-carrageenan, a mixture of kappa- andlambda-carrageenan, or a mixture of iota-, kappa- and lambda-carrageenanhomo- or heteropolymers, unless explicitly stated otherwise or unless adifferent meaning is derivable from the spirit of the pertinentdisclosure.

A carrageenan homopolymer is a molecularly pure carrageenan compound ofone type of either iota-, kappa- or lambda-carrageenan. A carrageenanheteropolymer comprises subunits of at least two different kinds ofcarrageenans, preferably selected from the group consisting of iota-,kappa- and lambda-carrageenan subunits.

Where referred to hereinafter, the term “mixture” of carrageenans mayalso refer to a composition of matter comprising as an active antiviralingredient at least one kind of carrageenan heteropolymer, the “mixture”thus primarily being a mixture of different carrageenan subunits as partof said at least one heteropolymeric carrageenan present in thecomposition.

In a further embodiment the invention relates to such an anti-rhinoviralcomposition for prophylactic or therapeutic use, wherein said rhinovirusinfection is an acute or a chronic rhinovirus infection.

The present carrageenan-based composition is suitable for topicalapplication to treat skin or mucosal inflammation. But also systemic,e.g. parenteral or oral application is possible, especially when adaptedto contain primarily low molecular weight carrageenan fractions. Thecarrageenan useful in the present invention has a mean molecular weightranging from about 15000 to 5000000 Da. The low molecular weightfraction comprises carrageenan at an average molecular weight rangingfrom about 15 000 to about 50 000 Da, the middle molecular weightfraction from about 50 000 to about 500 000 Da, and the high molecularweight fraction from about 500 000 to about 5 000 000 Da.

In a preferred embodiment the pharmaceutical composition is adapted fortopical or mucosal use. Suitable galenic forms of the ready-for-usepreparations are creames, gels, ointments, powders (including powdersfor inhalation), sprays, foams, or liquid solutions such as skinlotions, gargle solutions or nose drops. Other suitable forms of galenicpreparation will be evident to those of ordinary skill in the art,including, for example, the nasal drug delivery systems disclosed inU.S. Pat. No. 6,391,452.

Apart from the active antiviral ingredients the present compositiontypically comprises at least one pharmaceutically acceptable carrier,and optionally further additives or ingredients.

A suitable carrier may be a diluent, e.g. water or saline, an excipient,or another vehicle suitable and useful for the administration of theactive ingredients. Optional additives may be selected from the groupconsisting of SiO₂, TiO₂, a binder such as microcrystalline cellulose,polyvinylpyrrolidone, gum tragacanth, gelatine, starch, lactose, lactosemonohydrate, alginic acid or maize; a lubricant or surfactant such asmagnesium stearate or sodium lauryl sulphate; a glidant, such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin. Further additives in the preparation can be but are notlimited to buffers or pH adjusting agents, e.g. selected from citricacid, acetic acid, fumaric acid, hydrochloric acid, malic acid, nitricacid, phosphoric acid, propionic acid, sulfuric acid, tartaric acid, orcombinations thereof.

Further ingredients may be present, including non-carrageenan drugs orpharmaceutically active substances.

Carrageenan may be used in the form of any pharmaceutically acceptablesalt, for example sodium salts of carrageenan may be used. Otherpharmaceutically acceptable salts include, among others, potassium,lithium and ammonium salts of carrageenan.

In another embodiment the invention the composition is for topical useand comprises carrageenan in an amount of between 0.01% and 20%,preferably between 0.1% and 10%, most preferably between 0.5% and 5% byweight (w/w) of the preparation.

Usually, the composition will be provided as a non-pyrogenous, sterilepreparation. In case of a liquid preparation sterility may be achieved,for example, by filtration through a suitable membrane filter. Methodsfor the manufacture of sterile or aseptic pharmaceutical compositionsare well known in the art and are not part of the present invention.

However, the pharmaceutical composition of the present invention mayalso be coated onto solid surfaces of hygiene or sanitary items, forexample facial hygiene or sanitation articles that are typically used inthe oral and/or nasal areas such as nasal tissues or papers, andhandkerchiefs. More specifically, the pharmaceutical composition may beapplied, e.g. sprayed—much like disinfectants—onto gloves, hygienetissues or papers including nasal tissues, in order to exert a virucideeffect at least to some extent, thus contributing to reducing anindividual's repeated self-infection by contaminated fingertips and alsoto reduce viral spread among different individuals that are in close,e.g. hand-to-hand, contact with each other. Depending on the nature ofthe sanitary or hygiene item, the item may be covered, wet, or otherwiseimpregnated with the pharmaceutical composition.

Such carrageenan-treated items may also include but are not restrictedto cotton swabs, dust masks or facial masks. Even lipsticks maybeformulated to contain an antiviral effective amount of carrageenan.These hygiene or sanitation articles can be used prophylactically oralong with therapeutical treatment against a viral infection and mayassist in the prevention or reduction of a risk of infection.

Accordingly, in one embodiment the invention relates to such a use,wherein the antiviral composition is applied to the solid surface of ahygiene or sanitation article, particularly of a hygiene or sanitaryglove, tissue or paper, especially a nasal tissue or paper, by eithercoating or impregnation.

The iota-, kappa- and lambda-carrageenans useful in the presentinvention are commercially available but may also be prepared byextraction from seaweed plants pursuant to extraction procedures knownin the art.

In its preferred embodiment the invention relates to the use of at leastone member selected from the group consisting of the homo- andheteropolymers of iota-, kappa-, and lambda-carrageenan.

In a specific embodiment the antiviral pharmaceutical composition of thepresent invention is substantially free of carrageenan forms other thaniota-, kappa-, and lambda-carrageenan, —although trace amounts of suchother carrageenans may be present. For various applications,iota-carrageenan may substantially be the only kind of carrageenanpresent in the composition.

In another embodiment the invention relates the use of carrageenan inthe manufacture of an antiviral composition, wherein the compositioncomprises either iota-, kappa- or lambda-carrageenan, or a mixture of atleast two of said carrageenans, in an amount of 80% or more, 90% ormore, 95% or more, or even 99% or more of all carrageenans present inthe composition. The percentages are given in percent by weight (% w/w)relative to the dry weight of the carrageenans referred to.

In another embodiment the invention relates to such a use, wherein thecomposition comprises not less than 50%, not less than 70%, not lessthan 80%, and preferably not less than 95% (w/w) by dry weight ofiota-carrageenan, relative to the total dry weight of all carrageenanspresent in the composition.

The above carrageenan concentration values likewise apply to homo- andheteropolymeric carrageenans.

Carrageenan was found to be non-toxic upon oral or dermaladministration, or upon inhalation, even when applied at extremely highdoses and was therefore classified as “generally recognized as safe”(GRAS) by the Food and Drug Administration (FDA).

In another embodiment, the invention relates to the use of carrageenansin the manufacture of an antiviral composition, which further comprisesat least one additional pharmaceutically active, antiviral compound,preferably cellulose sulfate.

In another embodiment, the present invention relates to the use ofcarrageenan, preferably iota-carrageenan, in the manufacture of apharmaceutical composition for prophylactic or therapeutic treatment ofa bodily condition selected from the group consisting of microbialinfection, inflammatory disease, allergy, and impaired or suppressedimmune system, wherein the carrageenan is present in combination with atleast one other pharmaceutically active compound or drug. In such acomposition the carrageenan may exert anti-rhinoviral adjuvant function.Said at least one other pharmaceutically active compound or drug may beselected from the group consisting of a steroid, e.g. cortisone, and anantihistamine.

In another embodiment the antiviral pharmaceutical preparation is forthe treatment or prophylaxis in an individual that is especiallysusceptible to or is at an increased risk of developing into a high-riskpatient that is caused by a rhinovirus infection, such as a high-riskpatient selected from the group consisting of an asthma patient, aperson with allergies, and a person having an inflammatory disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a HRV induced cell death inhibition assay(XTT-assay).

-   -   Ordinate=OD measured at 492 nm; abscissa=different test samples;        1=uninfected cells; 2=untreated, infected cells; 3=infected        cells treated with iota-carrageenan; 4=infected cells treated        with kappa-carrageenan; 5=infected cells treated with        lambda-carrageenan; FIG. 1A=cells infected with HRV-2; FIG.        1B=cells infected with HRV-14.

FIG. 2 shows the determination of peak titers of supernatants frominfected Hela cells by TCID₅₀ assays.

-   -   Ordinate=TCID₅₀ titer; abscissa=concentration of        iota-carrageenan in μg/ml; U=untreated cells; T        (treatment)=cells were infected for one hour and the indicated        concentration of iota-carrageenan was added one hour after        treatment; P (prophylaxis)=the virus suspension was preincubated        with the indicated concentration of iota-carrageenan for on hour        before infection. Further processing was identical to T: cells        were infected at a multiplicity of infection of 0.01. The peak        titers were observed at day 3 for HRV-2 (FIG. 2A) and at day 4        for HRV-14 (FIG. 2B).

FIG. 3 shows the efficacy of iota-carrageenan on inhibition ofrhinovirus replication on infected human nasal epithelial cellsdetermined by TCID₅₀ assays.

-   -   Ordinate=TCID₅₀ titer in logs; abscissa=concentration of        iota-carrageenan in μg/ml; MOCK=untreated control cells; FIG.        3A=cells infected with HRV-1A; FIG. 3B=cells infected with        HRV-2; FIG. 3C=cells infected with HRV-8; FIG. 3D=cells infected        with HRV-16; FIG. 3E=cells infected with HRV-39; FIG. 3F=cells        infected with HRV-83.

FIG. 4 shows the efficacy of iota-carrageenan against rhinovirus afterrepeated treatment determined by CPE reduction assay.

-   -   Ordinate=CPE reduction in %; abscissa=concentration of        iota-carrageenan in μg/ml; Strain HRV2P0=original rhinovirus        strain (no replication round); Strain HRV2P10=rhinovirus strain        HRV2P0 after 10 selective replication rounds on HeLa cells.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. The examples are forillustrative purposes only and are not to be construed as limiting thisinvention in any respect. It is further understood that the presentinvention shall also comprise variations of the expressly disclosedembodiments to an extent as would be contemplated by a person ofordinary skill in the art.

Example 1: Effect of Different Types of Carrageenans Against HumanRhinovirus Type 2 (HRV-2) and Type 14 (HRV-14)

Subconfluent HeLa cells were incubated with a virus suspension that waspreincubated for 5 min with 125 μg/ml of the polymers as indicated inFIG. 1. 48 hours later the viability of the cells was determined withTOX2 XTT assay (Sigma).

As shown in FIG. 1, it was found that the most effective polymer wasiota-carrageenan (column 3) which was effective against both types ofrhinovirus while, lambda-carrageenan (column 5) and kappa-carrageenan(column 4) showed efficacy against HRV-2 but not against HRV-14. Errorbars indicate the standard deviation between six independent wells.

Example 2

The polymers indicated in Table 1 were tested in a HRV-2 and HRV-14induced cell death inhibition assay (XTT-assay) at a concentration of100 μg/ml.

As shown in Table 1, iota-carrageenan yielded protection against bothtypes of rhinovirus (a “+” indicates at least 95% protection as comparedwith uninfected control cells). Kappa-carrageenan and lambda-carrageenanwere active against HRV-2 but not against HRV-14. The polymers chitosan,carboxymethyl cellulose and carboxymethyl chitosan did not show aninhibitory effect at all.

TABLE 1 Antiviral activity of several tested polymers Polymer HRV-2HRV-14 Iota-Carrageenan + + Kappa-Carrageenan + − Lambda-Carrageenan + −Chitosan − − Carboxymethylcellulose − − Carboxymethylchitosan − −

Example 3

Iota-carrageenan was effective against HRV-2 and HRV-14 in a treatmentand a prophylaxis viral replication model. A significant reduction ofpeak viral titer was observed at concentrations equal and higher than6.25 μg/ml. However, iota-carrageenan was most effective in theprophylaxis model against HRV-2 in which a reduction of more than 99.9%in the peak viral titer was observed.

Example 4: Effect of Iota-Carrageenan Against a Selection of HumanRhinovirus Subtypes

Subconfluent HeLa cells in 96 well plates were incubated with a virussuspension at a MOI (multiplicity of infection) of 0.5.

20 min after infection with the virus suspension, nutrient mediumcontaining polymer in 3-fold dilutions was added. 48-72 hours later theviability of the cells was determined with TOX2 XTT assay (Sigma). EC₅₀values were calculated with the software Excel-Fit.

TABLE 2 Virus HRV-1 HRV-2 HRV-8 HRV-14 HRV-16 HRV-39 EC₅₀ 0.7 21 <0.5400 381 117 μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml

Iota-carrageenan was active against all tested human rhinoviruses onpreviously infected HeLa cells. Carrageenan concentrations needed toinhibit 50% of the cytopathic effect vary within a wide range of <0.5μg/ml for HRV-8 and 400 μg/ml for HRV-14. This result indicates thatiota-carrageenan inhibits the replication of a broad spectrum ofrhinovirus subtypes on infected HeLa cells.

Example 5: Inhibition of Rhinovirus Replication on Human NasalEpithelial Cells by Iota-Carrageenan

Primary human nasal epithelial cells (Promocell) were seeded in 24 wellplates (2.9*10⁴ cells per well) and cultivated for three days at 37° C.,5% CO₂ and 95% humidity. The cells were infected at a confluency ofnearly 60% with rhinovirus strains HRV-1A, HRV-2, HRV-8, HRV-14, HRV-16,HRV-39, HRV-83 and HRV-84. Rhinoviruses HRV-1A, HRV-2, HRV-8, HRV-16,HRV-39 and HRV-83 performed a lytic replication on human nasalepithelial cells. HRV-14 and HRV-84 did not lyse the cells and weretherefore not subjected to further testing. Viruses were preincubatedwith iota-carrageenan at concentrations of 4, 40 and 400 μg/ml and aMOCK control. The MOI was 0.34. Supernatants were harvested between 48and 72 hours after infection and used for TCID₅₀ titer determination. Asshown in FIG. 3, treatment with iota-carrageenan reduced the viral titerof all viruses in the supernatants at least two log steps at aconcentration of 40 μg/ml (>99%) when compared to MOCK treated controlcells (the Y-axis shows the virus titer in logs). These data clearlydemonstrate that iota-carrageenan inhibits the virus replication onprimary human epithelial cells. Since it appears that at present thereis no animal model for human rhinovirus in vivo testing available, thehuman nasal epithelial cells used herein represent the most important invitro model currently available. Error bars indicate the standarddeviation between three independent tested samples.

Example 6: Determination of the Efficacy of Iota-Carrageenan AgainstRhinovirus Infection after Repeated Treatment (Check for PossibleDevelopment of Viral Resistance)

The original virus HRV2P0 and the virus HRV2P10, obtained after 10selective replication rounds on HeLa cells, were tested in a CPE(cytopathic effect) reduction assay. HeLa cells (8*10⁴ cells per well)were seeded in six well plates. The cells were infected with a suitablenutrient medium supplemented with carrageenan polymer at finalcarrageenan concentrations of 1.6, 5.3, 17, 50, 150 and 450 μg/ml, andfurther containing virus at a MOI of 0.1. As controls, one well was MOCKinfected with virus-free, carrageenan-supplemented nutrient medium, andanother one well was infected with a MOCK treated virus suspension, i.e.a virus suspension containing nutrient medium but no carrageenan. Afteran incubation time of 20 minutes following infection, the plates werewashed twice and control medium (virus-free, supplemented withcaraageenan at the selected concentrations) was added to all wellsexcept to the mock-treated negative control (i.e. pure, uininhibitedinfection). Samples were taken from each experimental well as soon as aclear cytopathic effect was visible in the unprotected, negative controlwell. For the subsequent selection round, virus-containing supernatantsampled from those carrageenan-treated wells exhibiting a clearlydetectable CPE relative to the uninfected control was used for infectionof Hela cells. Typically, CPE responses were detectable in theexperimental samples at the two lowest carrageenan concentrations only,i.e. at 1.6 and 5.3 μg carrageenan per 1 ml.

The procedure was repeated 10 times and the resulting virus samples werecompared in a CPE inhibition test with the original virus stock.Briefly, 48 hours after infection a TOX2 reagent (Sigma) was added,OD_(450 nm) values were determined and the CPE reduction in % of theuninfected control was determined.

In order to check for possible development of virus resistance towardscarrageenan treatment, the virus used for the first inoculation (virus2_001 in FIG. 4) and a virus obtained after 10 selective replicationrounds (virus 20_001 in FIG. 4) were tested in a CPE reduction assayusing HeLa cells, following an experimental protocol that was slightlydifferent from the one described above. Briefly, HeLa cells wereinfected with the respective virus at a MOI of 0.1, and 20 minutes afterinfection the inoculum was removed and nutrient medium containingcarrageenan polymer at final concentrations of 1.6, 5.3, 17, 50, 150 and450 μg/ml was added (see abscissa of FIG. 4). 48 hours after infection aTOX2 reagent (Sigma) was added, OD450 nm values were determined and theCPE reduction in % of unifected control was determined (FIG. 4,ordinate). Error bars indicate the standard deviation between 6independent wells.

As graphically represented in FIG. 4, no significant difference in virussusceptibility towards carrageenan treatment was detected in the samplesobtained from the supernatant of the first and the tenth infectioncycle. This indicates that no escape mutants emerged during theselection procedure. These data thus suggest that an escape ofrhinoviruses during in vivo therapy with iota-carrageenan may beunlikely even in cases of continued or repeated therapy over an extendedperiod of time.

The invention claimed is:
 1. A method of reducing the risk of acquiringa rhinovirus infection in a subject in need thereof, the methodcomprising administering to the subject a pharmaceutical compositioncomprising, as the sole active antiviral ingredient, an iota-carrageenanhomopolymer and/or heteropolymer in an antiviral effective amount,wherein the administration is carried out topically by application toeither of, or to both of, the skin and nasal mucosa, and wherein thecomposition further comprises at least one non-carrageenanpharmaceutically active compound selected from the group consisting of asteroid and an antihistamine.
 2. The method according to claim 1,wherein the subject is in need of a reduction of the risk of acquiringan acute or chronic rhinovirus infection, and the subject suffers fromat least one disease selected from the group consisting of asthma,chronic obstructive pulmonary disease, cystic fibrosis, allergy, andinflammatory disease.
 3. The method according to claim 1, wherein thecomposition comprises the iota-carrageenan homopolymer and/orheteropolymer in an amount of between 0.01% and 20% (w/w) of thecomposition.
 4. A method of reducing the risk of acquiring a rhinovirusinfection in a subject in need thereof, the method comprisingadministering to the subject a pharmaceutical composition comprising, asthe sole active antiviral ingredient, an iota-carrageenan homopolymerand/or heteropolymer in an antiviral effective amount, wherein theadministration is carried out topically by application to either of, orto both of, the skin and nasal mucosa, and wherein the subject is anindividual being at high-risk of developing into a patient selected fromthe group consisting of a person suffering from allergy, and a personsuffering from an inflammatory disease.
 5. The method according to claim4, wherein the composition comprises the iota-carrageenan homopolymerand/or heteropolymer in an amount of between 0.01% and 20% (w/w) of thecomposition.
 6. A method of reducing the risk of acquiring a rhinovirusinfection in a subject that suffers from at least one disease selectedfrom the group consisting of asthma, chronic obstructive pulmonarydisease, cystic fibrosis, allergy, and inflammatory disease, the methodcomprising administering to the subject a pharmaceutical compositioncomprising, as the sole active antiviral ingredient, an iota-carrageenanhomopolymer and/or heteropolymer in an antiviral effective amount,wherein the administration is carried out either systemically by oralingestion, or topically by application to the skin and/or to the oraland/or nasal mucosa, or the administration is carried out bothsystemically by oral ingestion and topically by application to the skinand/or to the oral and/or nasal mucosa.
 7. The method according to claim6, wherein the composition further comprises at least onepharmaceutically acceptable carrier or additive.
 8. The method accordingto claim 6, wherein the composition comprises the iota-carrageenanhomopolymer and/or heteropolymer in an amount of between 0.01% and 20%(w/w) of the composition.
 9. The method according to claim 8, whereinthe composition comprises iota-carrageenan homopolymer and/orheteropolymer in an amount of between 0.1% and 10% (w/w) of thecomposition.
 10. The method according to claim 8, wherein thecomposition comprises iota-carrageenan homopolymer and/or heteropolymerin an amount of between 0.5% and 5% (w/w) of the composition.
 11. Themethod according to claim 6, wherein the iota-carrageenan heteropolymercomprises at least one other carrageenan selected from kappa-carrageenanand lambda-carrageenan.
 12. The method according to claim 6, whereinprior to the administering, the composition is coated onto a solidsurface of a hygiene or sanitation article, and the administeringcomprises the subject contacting the coated surface of the hygiene orsanitation article.
 13. The method according to claim 12, wherein thecomposition is coated onto a solid surface of a hygiene or sanitaryglove, tissue or paper.
 14. The method according to claim 12, whereinthe composition is coated onto a solid surface of a nasal tissue orpaper.
 15. The method according to claim 6, wherein the composition isadministered as a nose spray, a powder, a gel, an ointment, a foam, or aliquid solution.
 16. The method according to claim 6, wherein thecomposition comprises not less than 95% (w/w), based on weight, ofiota-carrageenan relative to a total of all carrageenans present in thecomposition.
 17. The method according to claim 6, wherein the subject isa child.
 18. The method according to claim 6, wherein the rhinovirusinfection is an acute or a chronic rhinovirus infection.
 19. The methodaccording to claim 6, wherein the composition comprises not less than80% (w/w), based on weight, of iota-carrageenan relative to a total ofall carrageenans present in the composition.
 20. The method according toclaim 6, wherein the composition further comprises at least onenon-carrageenan pharmaceutically active compound selected from the groupconsisting of a steroid and an antihistamine.